Abstract
Polyadenylation of nascent RNA by poly(A) polymerase (PAP) is important for 3′ end maturation of almost all eukaryotic mRNAs. Most mammalian genes harbor multiple polyadenylation sites (PASs), leading to expression of alternative polyadenylation (APA) isoforms with distinct functions. How poly(A) polymerases may regulate PAS usage and hence gene expression is poorly understood. Here, we show that the nuclear canonical (PAPα and PAPγ) and non-canonical (Star-PAP) PAPs play diverse roles in PAS selection and gene expression. Deficiencies in the PAPs resulted in perturbations of gene expression, with Star-PAP impacting lowly expressed mRNAs and long-noncoding RNAs to the greatest extent. Importantly, different PASs of a gene are distinctly regulated by different PAPs, leading to widespread relative expression changes of APA isoforms. The location and surrounding sequence motifs of a PAS appear to differentiate its regulation by the PAPs. We show Star-PAP-specific PAS usage regulates the expression of the eukaryotic translation initiation factor EIF4A1, the tumor suppressor gene PTEN and the long non-coding RNA NEAT1. The Star-PAP-mediated APA of PTEN is essential for DNA damage-induced increase of PTEN protein levels. Together, our results reveal a PAS-guided and PAP-mediated paradigm for gene expression in response to cellular signaling cues.
Highlights
Most eukaryotic mRNAs employ cleavage and polyadenylation (CPA) for 3 end maturation [1,2]
Using quantitative real-time RTPCR (qRT-polymerase chain reaction (PCR)) and IB, we found that etoposide treatment increased PTEN protein levels in dose- and time-dependent manners (Supplementary Figure S10A and B), which corresponded to enhanced PTEN mRNA expression (Figure 5D). siStar-poly(A) polymerase (PAP) decreased the basal PTEN levels as shown in Figure 5B and ablated the DNA damage-augmented PTEN mRNA and protein expression (Figure 5D)
polyadenylation sites (PASs) regulation by PAPs involves both common and unique 3 end processing factors that are modulated by upstream generic transactivators in response to cellular signals, resulting in alternative polyadenylation (APA) or/and changes in the expression levels of gene transcripts
Summary
Most eukaryotic mRNAs employ cleavage and polyadenylation (CPA) for 3 end maturation [1,2]. The machinery responsible for CPA includes several subcomplexes [5], such as the Cleavage and Polyadenylation Specificity Factor (CPSF), comprising CPSF-160, CPSF100, CPSF-73, CPSF-30, Fip and WDR33; the Cleavage Stimulation Factor (CstF), comprising CstF-50, CstF64/CstF-64 and CstF-77; the Cleavage Factor (CF) I, comprising CFI-25, CFI-59 and CFI-68; CF II, comprising Pcf and Clp; and a number of single proteins, such as symplekin, nuclear poly(A) binding protein (PABPN1) and poly(A) polymerase (PAP). Three canonical nuclear PAPs have been identified in mammalian cells, including PAP␣ and PAP␥ /neo-PAP, both of which are ubiquitously expressed PAPs [6,7], and PAP, whose expression appears to be restricted to testis [8]. Previous studies have shown that Star-PAP directly binds target pre-mRNAs upstream of the PAS and recruits CPSF for 3 end cleavage [10,12].
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